PROJECT SUMMARY In the United States, millions of people suffer from glaucoma and atypical optic neuritis, which causes irreversible blindness due to the death of retinal ganglion cells (RGCs) and their projecting axons. For the past decade, the field has focused on promoting RGC survival and axonal regeneration. Despite great strides in these two areas, we have uncovered a new hurtle to restoring these neural circuits: regenerating RGC axons are not myelinated. Myelin is essential for efficient axonal conduction, and it provides important support for the long-term survival of regenerating axons. This myelination failure represents an underappreciated and essential aspect for the restoration of functional vision recovery. Oligodendrocyte precursor cells (OPCs) are a progenitor population that exist within the central nervous system (CNS), and are responsible for the generation of myelination-competent oligodendrocytes throughout adulthood. However, our regeneration data shows that the presence of axons alone is not sufficient to elicit myelination, and suggests the existence of an extrinsic barrier to OPC maturation. Using the mouse optic nerve crush (ONC) model, we found that chronically activated microglia inhibit the maturation of OPCs into myelinating oligodendrocytes. The elimination of microglia after ONC, using pharmacological methods, allows for an unprecedented rate of myelination. However, premature elimination of microglia, prior to ONC, results in a loss of OPC proliferation and is severely detrimental to myelination. Therefore, activated microglia are simultaneously responsible for promoting and inhibiting distinct aspects of OPC development after injury. To better understand this phenomenon, I will conduct single cell RNA sequencing (Smart-Seq2) on OPC lineage cells after ONC, and in the presence or absence of activated microglia. I can then examine the rate of oligodendrocyte maturation, and the effect of extrinsic inhibition from microglia on the transcriptomic profiles of OPC lineage cells. In addition, I will sequence activated microglia after ONC to identify candidate molecules that, when inhibited, could increase oligodendrocyte maturation and myelination. Candidate molecules will be examined in vivo using viral vector knockdown strategies to test their efficacy in promoting the myelination of regenerating axons after injury. This project holds the potential to identify new signaling pathways between activated microglia and maturing oligodendrocytes that prevent the myelination of axons in an inflammatory context. These findings will provide key insights for vision restoration after optic nerve injury or disease, and they could also help promote the remyelination of axons in patients suffering from other CNS disorders, such as multiple sclerosis and leukodystrophies. Dr. Zhigang He's lab and the F.M. Kirby Neurobiology Center at Boston Children's Hospital are incredibly collegial and well-equipped institutions that will suppor...